Rolling Circle Amplification in a Prokaryotic Translation System Using Small Circular RNA
Rolling Circle Amplification in a Prokaryotic Translation System Using Small Circular RNA
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DOI：
10.1002/anie.201302044
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Abe, Hiroshi
中科院分区：
文献类型：
--
作者：
Abe, Naoko;Hiroshima, Michio;Abe, Hiroshi
滚环扩增(RCA)是一种由一组特定的DNA聚合酶介导的等温、酶促过程，在此过程中，在一个短的环状单链DNA分子上合成重复的单链DNA分子(图1a)。[1]该反应产生长核酸，因为环状模板本质上代表一个无限长的模板。发现后，RCA最初被用于超灵敏的DNA检测。[1]最近，该技术还被用于检测核酸以外的目标分子，如小分子和蛋白质。[1]在RCA中，长度为13个核苷酸(NT)的环状单链DNA分子被证明是聚合酶的底物。[2]RCA的关键特征之一是，它的机制不仅提供了长重复寡核苷酸的途径，而且还在给定的时间段内提供了更高的产量水平。通过避免聚合酶和模板之间的多重结合和解离过程，RCA有效地促进了寡核苷酸的合成。[3]1998年，Perriman和Ares证明了环状RNA分子可以在大肠杆菌中活体翻译。[4]他们研究的主要目的是构建一个在大肠杆菌中产生重复蛋白序列的系统。当在细胞中形成具有无限开放阅读框架(ORF)的795个核苷酸的RNA环时，在大肠杆菌中产生了长链的多聚体绿色荧光蛋白(GFP)，尽管他们认为重复的GFP不发出荧光。[4A]在这项开创性的研究中，得出结论：环状RNA在翻译过程中提供的产物比线性RNA少，因为环状RNA的启动效率低于线性RNA。[4A]然而，在这份特殊的报告中，他们没有比较当连续的蛋白质合成发生时蛋白质产量。原则上，当
Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by a specific group of DNA polymerases in which tandemly repeated single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA molecule (Figure 1a).[1] The reaction produces long nucleic acids because the circular template in essence represents a template of infinite length. Following its discovery, RCA was initially used for the ultrasensitive detection of DNA.[1] More recently, the technique has also been used to detect target molecules other than nucleic acids, such as small molecules and proteins.[1] In RCA, circular ssDNA molecules as short as 13 nucleotides (nt) in length have been shown to act as substrates for polymerases.[2] One of the key features of RCA is that its mechanism not only provides access to long repeating oligonucleotides but that it also provides enhanced levels of production over a given period of time. The synthesis of oligonucleotides by RCA is effectively promoted by the avoidance of the multiple association and dissociation processes between the polymerase and the template.[3] In 1998, Perriman and Ares demonstrated that circular RNA molecules could be translated in vivo in E. coli.[4] The principle aim of their study was the construction of a system to produce repeating protein sequences in E. coli. A long chain of multimeric green fluorescent protein (GFP) was produced in E. coli when an RNA circle of 795 nt with an infinite open reading frame (ORF) was formed in the cell, although they suggested that the repeating GFP did not emit fluorescence.[4a] In this pioneering study, it was concluded that the circular RNA provided a smaller amount of product from the translation process than linear RNA, because initiation was less efficient on the circular RNA than on the linear RNA.[4a] In this particular report, however, they did not compare the amount of protein production when the continuous protein synthesis occurred. In principle, when the